Identification of BAY61-3606 Derivatives With Improved Activity in Splicing Modulation That Induces Inclusion of Cassette Exons Similar to the Splicing Factor 3B Subunit 1 Mutation.

Splicing modulation by a small compound offers therapeutic potential for diseases caused by splicing abnormality. However, only a few classes of compounds that can modulate splicing have been identified. We previously identified BAY61-3606, a multiple kinase inhibitor, as a compound that relaxes the splicing fidelity at the 3' splice site recognition. We have also reported the synthesis of derivatives of BAY61-3606. In this study, we tested those compounds for their splicing modulation capabilities and identified two contrasting compounds. These compounds were further investigated for their effects on the whole transcriptome, and analysis of changes in transcription and splicing revealed that the highly active derivative in the splicing reporter assay also showed significantly higher activity in modulating the splicing of endogenously expressed genes. Particularly, cassette exon inclusion was highly upregulated by this compound, and clustering analysis revealed that these effects resembled those in splicing factor 3b subunit 1 (SF3B1) K700E mutant cells but contrasted with those of the splicing inhibitor H3B-8800. Additionally, a group of serine/arginine-rich (SR) protein genes was identified as representatively affected, likely via modulation of poison exon inclusion. This finding could guide further analysis of the mode of action of these compounds on splicing, which could be valuable for developing drugs for diseases associated with splicing abnormalities.

Curcumin degradation in a soil microorganism: Screening and characterization of a ß-diketone hydrolase.

Curcumin is a plant-derived secondary metabolite exhibiting antitumor, neuroprotective, antidiabetic activities, and so on. We previously isolated Escherichia coli as an enterobacterium exhibiting curcumin-converting activity from human feces, and discovered an enzyme showing this activity (CurA) and named it NADPH-dependent curcumin/dihydrocurcumin reductase. From soil, here, we isolated a curcumin-degrading microorganism (No. 34) using the screening medium containing curcumin as the sole carbon source and identified as Rhodococcus sp. A curcumin-degrading enzyme designated as CurH was purified from this strain and characterized, and compared with CurA. CurH catalyzed hydrolytic cleavage of a carbon-carbon bond in the ß-diketone moiety of curcumin and its analogs, yielding two products bearing a methyl ketone terminus and a carboxylic acid terminus, respectively. These findings demonstrated that a curcumin degradation reaction catalyzed by CurH in the soil environment was completely different from the one catalyzed by CurA in the human microbiome. Of all the curcumin analogs tested, suitable substrates for the enzyme were curcuminoids (i.e., curcumin and bisdemethoxycurcumin) and tetrahydrocurcuminoids. Thus, we named this enzyme curcuminoid hydrolase. The deduced amino acid sequence of curH exhibited similarity to those of members of acetyl-CoA C-acetyltransferase family. Considering results of oxygen isotope analyses and a series of site-directed mutagenesis experiments on our enzyme, we propose a possible catalytic mechanism of CurH, which is unique and distinct from those of enzymes degrading ß-diketone moieties such as ß-diketone hydrolases known so far.

Divergent synthesis of kinase inhibitor derivatives, leading to discovery of selective Gck inhibitors.

We accomplished divergent synthesis of potent kinase inhibitor BAY 61-3606 (1) and 27 derivatives via conjugation of imidazo[1,2-c]pyrimidine and indole ring compounds with aromatic (including pyridine) derivatives by means of palladium-catalyzed cross-coupling reaction. Spleen tyrosine kinase (Syk) and germinal center kinase (Gck, MAP4K2) inhibition assays showed that some of the synthesized compounds were selective Gck inhibitors.